Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology

6

Weeks in Review

2

Weeks to Publication
Indexed in KCI, KoreaMed, Synapse, DOAJ
Open Access, Peer Reviewed
pISSN 2288-0585 eISSN 2288-6850

Analysis of Enterovirus Genotypes Using Single-Strand Conformational Polymorphism

Original article

Annals of Clinical Microbiology (Ann Clin Microbiol) 2001 December Volume 4, Issue 2, pages 134-138.

Analysis of Enterovirus Genotypes Using Single-Strand Conformational Polymorphism

Jung-Hyun Kim*,**, Jung-Hee Lee*, Je-Kue Lee*, Won-Ki Bang**, Jung-Yon Hong***, and Eui-Chong Kim*,****

Virus Research Center, Clinical Research Institute, Seoul National University Hospital*; Graduate School of Natural resources, Korea University**; Department of Pediatrics, Jeju Medical Center***; Department of Clinical Pathology, Seoul National University College of Medicine****

Abstract

Background: Epidemics of aseptic meningitis due to enteroviruses has occurred annually in the late spring and early summer season. The early detection of such epidemics is important for the prevention of further infection. Enteroviruses consist of 67 serotypes but one or two serotypes may be the causative agents of epidemics in the year and several different serotypes involve sporadic cases. In order to discriminate epidemics from sporadic infection, the serotype should be determined. We evaluated the significance of the single-strand conformational polymorphism (SSCP) of reverse transcription-polymerase chain reaction (RT-PCR) products of 5’-untranslated region (UTR) for the determination of serotypes.

Methods: The specimens of patients were cultured with RD cell and the RT-PCR was performed in case of the positive cytopathic effect. For the amplification of 153-bp of 5’-UTR, primers (EN1: 5’-CTC CGG CCC CTG AAT GCG GCT AAT-3’; EN2: 5’-ATT GTC ACC ATA AGC AGC CA-3’) were used. The RT-PCR products were denatured with 95% formamide at 95℃for 5 min and SSCP was performed. 12.5% polyacrylamide gel (49/1 acrylamide/bis) was made by using Mighty SmallTM SE245 Dual Gel Caster (Hoefer Scientific Instruments Inc., USA). Electrophoresis was done at 10 mA for 1.5 h, and silver nitrate stain was performed. The SSCP patterns were compared with serotypes determined by sequence analysis of VP1 region.

Results : Coxsackievirus A9 (two strains), coxsackievirus A16 (10), coxsackievirus B2 (two), coxsackievirus B3 (two), echovirus 3 (two), echovirus 11 (two), echovirus 16 (seven), echovirus 19 (two), echovirus 30 (three), and enterovirus 71 (six) showed the different SSCP patterns according to their serotypes. The same pattern was observed in the same serotype, except echovirus 30 showing two different patterns.

Conclusions : The SSCP of RT-PCR products of 5’-UTR may be helpful to determine the serotype and discriminate epidemics from sporadic cases. This has the advantage to be able to test the specimens directly without the viral culture. But the serotype should be determined by other method such as neutralization or sequence analysis in case of the first isolate in the epidemic season and the stains showing the new SSCP patterns. (Korean J Clin Microbiol 2001;4(2):134-138)

Keywords

Bacteremia, Blood culture, Blood volume, Educational intervention, Quality improvement